A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no “gold standard” assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.     

Deviation from Clinical Routines Can Reveal Sources of Device Design Vulnerability

The ability to adequately ventilate a patient is critical and sometimes a challenge in the emergency, intensive care, and anesthesiology settings. Commonly, initial ventilation is achieved through the use of a face mask in conjunction with a bag that is manually squeezed by the clinician to generate positive pressure and flow of air or oxygen through the patient''s airway. Large or small erroneous openings in the breathing circuit can lead to leaks that compromise ventilation ability. Standard procedure in anesthesiology is to check the circuit apparatus and oxygen delivery system prior to every case. Because the face mask itself is not a piece of equipment that is associated with a source of leak, some common anesthesia machine designs are constructed such that the circuit is tested without the mask component. We present an example of a leak that resulted from complete failure of the face mask due to a tiny tear in its cuff by the patient''s sharp teeth edges. This subsequently prevented formation of a seal between the face mask and the patient''s face and rendered the device incapable of generating the positive pressure it is designed to deliver. This instance depicts the broader lesson that deviation from clinical routines can reveal unappreciated sources of vulnerability in device design.

Ventilation is the movement of air or gas from the external environment into the alveoli of the lungs. In the critical care setting, the ability to mechanically ventilate a patient in acute distress is a lifesaving skill, as these patients often cannot adequately breathe on their own. As such, ventilating is almost always more important than intubation per se. In emergencies, initial ventilation typically is established using a simple face mask in conjunction with a bag that the clinician manually squeezes to generate positive pressure and gas flow through the patient''s airway. These bags are commonly referred to as Ambu bags, a proprietary term that traces to a popular airway equipment brand. When a patient is breathing spontaneously, their inspiratory muscles, mainly the diaphragm, generate a pressure force that by convention is referred to as a “negative inspiratory force” or negative pressure that pulls outside air into the lungs. In contrast, when an inspiratory drive is absent, air or other gases can be “pushed” into the lungs by mechanical means (referred to clinically as positive pressure).Face masks are designed to have a soft-contoured, air-filled, cushion-like cuff that lies directly on the patient''s face, thereby allowing a seal to be formed over the mouth and nose (Figure 1). Achieving a proper seal is crucial to generating positive pressure. The cushion commonly consists of polyvinyl chloride plastisol due to its malleable properties.1 These masks are used extensively in anesthesiology because general anesthesia, and particularly intravenous (IV) anesthetics, often impede a patient''s ability to breathe on their own.Open in a separate windowFigure 1.Demonstration of a clinical face mask placement with the air-filled cuff cushion forming a seal around the face.Further, a paralytic medication is typically used in the setting of general anesthesia in order to optimize intubation conditions before an endotracheal tube is placed to secure the airway. The paralytic medication will completely prevent all skeletal muscle movements, including those of the diaphragm, hence eliminating any remaining spontaneous breathing drive that the patient may still have. The typical clinical sequence of events on induction (i.e., initiation of the anesthesia) of general anesthesia is to administer the IV anesthetic first and, only after adequate mask ventilation is confirmed, to then follow with a paralytic medication.In the event that mask ventilation fails (e.g., upper airway obstruction, equipment failure, or an unexpected airway pathology such as a tracheal fistula), the clinician may be able to backtrack to safety if a spontaneous respiratory drive is regained by the patient. Common IV induction agents such as propofol have the ideal pharmacokinetic property of a very short duration of action. Accordingly, their respiratory depressing effect can potentially be undone with the passage of time by opting to awaken the patient if ventilation cannot be achieved as expected2.Before each procedure, the standard of care in anesthesiology is to check the circuit apparatus as part of the anesthesia machine check.3 This machine check includes a positive pressure test whereby the breathing circuit is checked for leaks. The overall steps to the positive pressure test are outlined in Figure 2. Some anesthesia machines automatically perform the test on their own with the press of a button by the clinician. A common design in anesthesia machines includes a blank metal knob to which the circuit can be connected in order to close off the circuit and allow for pressurization by the machine (Figure 3). Such design requires intentional physical removal of the mask from the circuit in order to occlude the apparatus. The implicit assumption in this design is that the mask is reliable enough to be removed and excluded from the pressure test. This contrasts with the broad recognition that the mask is a vital component of the breathing circuit and without which the circuit is almost useless.Open in a separate windowFigure 2.Flowchart depicting the steps for performing a positive pressure test.Open in a separate windowFigure 3.Anesthesia machine circuit. Left: Face mask attachment connected. Right: Closed-off cap position for pressure test.We experienced failure of this type of mask and, at the time of the incident, were unable to identify its cause. After we induced general anesthesia, it was immediately evident that we could not generate positive pressure and failed to ventilate. After ruling out patient-specific causes that would interfere with ventilation, various external equipment-related reasons must be considered.The most common culprits for such a scenario are probably the presence of a leak somewhere along the path of oxygen flow or insufficient oxygen supply to the machine. These leaks can occur in the connections of the breathing circuit or within the anesthesia machine itself. At the time of our experience, we could not identify an apparent leak and the patient''s oxygen saturation rapidly desaturated as we were failing to ventilate adequately. Thankfully, intubation was successful, and ventilation was then established via the endotracheal tube.Afterwards, a close examination of the equipment eventually revealed a tear in the cuff of the mask. We believe that the mask was torn during induction when general anesthesia was initiated. This elderly patient had advanced dementia and refused to let the clinical team establish IV access. The decision was made to perform a mask induction in which inhaled anesthetic gases are used via the face mask instead of IV induction agents. This practice is common in young pediatric patients, for which IV access is challenging to achieve before induction of anesthesia. Given that our patient of this instance was elderly, he was missing numerous teeth and his remaining teeth were larger than those of a pediatric patient (Figure 4). When mask induction was initiated, the patient thrashed his head aggressively from side to side and grabbed the mask with his hands to forcefully remove it from his face. This necessitated the clinician to firmly hold the mask to the patient''s face. In this process, the sharp edges of the patient''s teeth likely caught the cuff and tore it.Open in a separate windowFigure 4.Actual patient''s dentition.Before this incident, we did not suspect the mask itself to be the equipment piece responsible for the leak. Given its exclusion from the pressure test, it is likely that the engineers who designed the anesthesia machine also did not think of it as a culprit for a leak. A leak around the mask is a common etiology of failed ventilation, but this occurs due to difficult airway features, such as a beard, deformed or abnormal facial structure, or conditions requiring considerably higher pressures to be generated (e.g., an obese patient).This patient''s airway was clinically unremarkable on exam during the preoperative physical evaluation. A leak around the mask was therefore not thought of and was low on the differential of problem etiology. Our general approach to diagnosing real-time leaks that occur after a proper machine check with a satisfactory pressure test was to focus on any changes that may have occurred after the test and to listen for audible signs of a leak. Before this incident, we partitioned leaks into two groups based on the physical size of the opening: large versus small equipment deformities. Our impression was that a large opening (e.g., a marked circuit disconnect) would be relatively obvious and visually apparent, whereas a small opening (e.g., tear, hole, loose connection fitting) would be more easily evident by an audible hissing sound. Our thought process was that a minor insult or opening in the circuit would still allow for some level of pressure to be generated within the apparatus and that this pressure escapes or leaks with turbulence and is therefore audible.As the above experience illustrates, the aforementioned dichotomy is a categorization not always maintained. The tear in the mask was tiny and on the inside portion of the mask (Figure 5), making it hard to visualize or hear. Nonetheless, because it was a tear in a cuff, it translated to a larger area of compromise that effectively prevented any positive pressure from being generated.Open in a separate windowFigure 5.Tear on the inside of the face mask cuff.This example demonstrates that even though the face mask itself does not take part in the positive pressure leak test, it can still be an important source of a major leak. Moreover, it highlights that when a medical device is used in a fashion at variance with its usual use or under altered conditions, extra vigilance to new sources of malfunction is warranted.     

Effective use of zinc oxide nanoparticles through root dipping on the performance of growth,quality, photosynthesis and antioxidant system in tomato

Journal of Plant Biochemistry and Biotechnology - The objective of the study was to explore the effect of zinc oxide nanoparticles (ZnO-NPs) through root treatment on growth biomarkers,...     

Precipitating and relieving factors of migraine versus tension type headache

Background

The correlation between intracranial pressure (ICP) and intraocular pressure (IOP) is still controversial in literature and hence whether IOP can be used as a non-invasive surrogate of ICP remains unknown. The aim of the current study was to further clarify the potential correlation between ICP and IOP.

Methods

The IOP measured with Goldmann applanation tonometer was carried out on 130 patients whose ICP was determined via lumber puncture. The Pearson correlation coefficient between ICP and IOP was calculated, the fisher line discriminated analysis to evaluate the effectivity of using IOP to predict the ICP level.

Results

A significant correlation between ICP and IOP was found. ICP was correlated significantly with IOP of the right eyes (p?<?0.001) and IOP of the left eyes (p?=?0.001) and mean IOP of both eyes (p?<?0.001), respectively. However, using IOP as a measurement to predict ICP, the accuracy rate was found to be 65.4%.

Conclusion

Our data suggested that although a significant correlation exists between ICP and IOP, caution needs to be taken when using IOP readings by Goldmann applanation tonometer as a surrogate for direct cerebrospinal fluid pressure measurement of ICP.     

The Primary Structure of β<Superscript>I</Superscript>-Chain of Hemoglobin from Snake Sindhi Krait (<Emphasis Type="Italic">Bungarus sindanus sindanus</Emphasis>)

The amino acid sequence of β^I-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of β^II, β^I, α^D and α^A-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of β^I globin chain from Sindhi Krait showed closest relationship with β^I globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of β^I globin chain of Sindhi Krait with human β chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αβ contact points and in regions directly not important for function.     

Kinetic characterization of recombinant human cystathionine β-synthase purified from E. coli

Cystathionine β-synthase (CBS) catalyzes the pyridoxal-5′-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence of a protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86–91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for l-Cth production, employing cystathionine β-lyase and l-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by l-Hcys (K_i^l-Hcys = 2.1 ± 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of l-Cth to l-Ser and l-Hcys were also determined and the k_cat/K_m^l-Cth of this reaction is only 2-fold lower than the k_cat/K_m^l-SER of the physiological, condensation reaction.     

Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.